Stable high-copy-number integration of Aspergillus oryzae alpha-amylase cDNA in an industrial baker's yeast strain

The Aspergillus oryzae alpha-amylase cDNA was placed under the control of t he Saccharomyces cerevisiae actin promoter (pACT1) and introduced into the ribosomal DNA locus of an industrial baker's yeast strain. To obtain a stra in eligible for commercial use, we constructed an integrative cassette lack ing bacterial DNA sequences but containing the alpha-amylase cDNA and ribos omal DNA sequences to target the integration to this locus. High-copy-numbe r integrants were obtained including a defective TRP1d promoter in the inte grative cassette. We selected one transformant, Rib-AMY (CECT10872), in whi ch the multi-integrated sequences were stable even after 200 generations of growth in nonselective medium. This transformant also expressed and secret ed high levels of alpha-amylase. Bread made with this strain had a higher: volume, lower density, and softer crumbs than bread made with a control str ain. The Rib-AMY transformant also was useful in retarding bread firming. T his new strain fulfills all the requirements for commercial utilization and should reduce or eliminate the requirement for addition of exogenous alpha -amylase to the flour, reducing allergenic work-related symptoms due to thi s enzyme.